Engineered biological systems hold potential in programming cell behavior to advance sustainable technologies, materials synthesis, and human health. However, incomplete understanding of the sequence-structure-function relationships that govern the design space limits our capacity to access, process, and act on information in living systems. Methods for assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are thus critical to advancing our ability to manipulate and interface with biology.
Programmable RNA-based gene-regulatory devices comprise parts that encode sensing, information transmitting, and actuating functions. RNA device architectures connect sensor and actuator components, such that sensor-detected information is transmitted into controlled activity of the actuator. One class of RNA devices utilizes a hammerhead ribozyme (HHRz) actuator to modulate the stability of a target transcript through conditional control of cleavage activity via binding of the cognate ligand. The ribozyme-based device framework supports genetic controllers in different organisms, responsive to diverse ligands, exhibiting complex computation, and applied to regulate complex phenotypes. Sensor and actuator components are linked through a rationally designed or screened transmitter that guides secondary structure changes in the components. As RNA folding is largely hierarchical and dictated by localized hydrogen bonding and base stacking, secondary structure changes are tractable. While this approach enables sequence-level modular device design, it limits regulatory potential. The relatively slow kinetics associated with the transmitter-induced secondary structure rearrangement places a limit on self-cleavage kinetics, over which a trade-off between gene-silencing activity and ligand sensitivity is observed. To address performance limitations inherent with secondary structure switching RNA devices, a new device architecture that achieves faster switching is needed.
High-throughput in vitro and in vivo selection and screening strategies for creating RNA devices have been described. In vitro selections have largely been supplanted by cell-based (in vivo) strategies to avoid any change in activities when transitioning from in vitro to in vivo environments. In vivo strategies link device activity to a readily measureable expression output, such as fluorescence, motility, or viability. These strategies only reveal sequence-activity information on a small number of individually-tested sequences. Strategies that provide sequence-activity information for all members in large libraries are needed to rapidly identify all high-functioning RNA devices and gain a complete understanding of the sequence-structure-function landscape to enable more robust design strategies. Methods that integrate fluorescence activated cell sorting (FACS) and high-throughput next generation sequencing (NGS) have been applied to investigate and/or develop gene-regulatory elements such as translation initiation sites, N-terminal codons, and various cis-regulatory elements.